Genomes and transcriptomes help unravel the complex life cycle of the blastoclad fungus, Coelomomyces lativittatus, an obligate parasite of mosquitoes and microcrustaceans.

Species of the phylum Blastocladiomycota, early-diverging zoosporic (flagellated) lineages of fungi, are vastly understudied. This phylum includes the genus Coelomomyces, which consists of more than 80 fungal species that are obligate parasites of arthropods. Known Coelomomyces species lack a complete asexual life cycle, instead surviving through an obligate heterecious alternation of generations life cycle. Despite their global distribution and interesting life cycle, little is known about the genomics of any Coelomomyces species. To address this, we generated three draft-level genomes and annotations for C. lativittatus representing its haploid meiospore, orange gamete, and amber gamete life stages. These draft genome assemblies ranged in size from 5002 to 5799 contigs, with a total length of 19.8-22.8 Mb and a mean of 7416 protein-coding genes. We then demonstrated the utility of these genomes by combining the draft annotations as a reference for analysis of C. lativittatus transcriptomes. We analyzed transcriptomes from across host-associated life stages, including infected larvae and excised mature sporangia from the mosquito Anopheles quadrimaculatus. We identified differentially expressed genes and enriched GO terms both across and within life stages and used these to make hypotheses about C. lativittatus biology. Generally, we found the C. lativittatus transcriptome to be a complex and dynamic expression landscape; GO terms related to metabolism and transport processes were enriched during infection and terms related to dispersal were enriched during sporulation. We further identified five high mobility group (HMG)-box genes in C. lativittatus, three belonging to clades with mating type (MAT) loci from other fungi, as well as four ortholog expansions in C. lativittatus compared with other fungi. The C. lativittatus genomes and transcriptomes reported here are a valuable resource and may be leveraged toward furthering understanding of the biology of these and other early-diverging fungal lineages.

mTORC2/Rictor is essential for coelomocyte endocytosis in Apostichopus japonicus.

Endocytosis plays an important role in the immune defence systems of invertebrates through the interaction between the mechanical target of rapamycin complex 2 (mTORC2) and the AGC kinase family. Rictor is the most important unique subunit protein of mTORC2 and is thought to regulate almost all functions of mTORC2, including endocytosis. In the present study, a novel invertebrate Rictor homologue was identified from Apostichopus japonicus (designated as AjRictor) via the rapid amplification of cDNA ends (RACE). Spatial expression analysis indicated that AjRictor is ubiquitously expressed in all the examined tissues and has the highest transcript level in coelomocytes. Vibrio splendidus challenge in vivo and lipopolysaccharide (LPS) exposure in vitro could remarkably up-regulate the messenger RNA (mRNA) expression of AjRictor compared with the control group. AjRictor knockdown by 0.49- and 0.69-fold resulted in the significant decrease in endocytosis rate by 0.53- (P < 0.01) and 0.59-fold (P < 0.01) in vivo and in vitro compared with the control group, respectively. Similarly, the treatment of coelomocytes with rapamycin for 24 h and the destruction of the assembly of mTORC2 markedly decreased the endocytosis rate of the coelomocytes by 35.92% (P < 0.05). We detected the expression levels of endocytosis-related molecular markers after AjRictor knockdown and rapamycin treatment to further study the molecular mechanism between mTORC2 and endocytosis. Our results showed that AGC kinase family members (PKCα and Pan1) and the phosphorylation level of AktS473 were remarkably decreased after reducing mTORC2 activity; thus, mTORC2/Rictor plays a key role in the immune regulation of endocytosis in coelomocytes. Our current study indicates that mTORC2/Rictor is involved in the coelomocyte endocytosis of sea cucumber and plays an essential regulation role in defending pathogen invasion.

Underwater high frequency noise: Biological responses in sea urchin Arbacia lixula (Linnaeus, 1758).

Marine life is extremely sensitive to the effects of environmental noise due to its reliance on underwater sounds for basic life functions, such as searching for food and mating. However, the effects on invertebrate species are not yet fully understood. The aim of this study was to determine the biochemical responses of Arbacia lixula exposed to high-frequency noise. Protein concentration, enzyme activity (esterase, phosphatase and peroxidase) and cytotoxicity in coelomic fluid were compared in individuals exposed for three hours to consecutive linear sweeps of 100 to 200 kHz lasting 1 s, and control specimens. Sound pressure levels ranged between 145 and 160 dB re 1µPa. Coelomic fluid was extracted and the gene and protein expression of HSP70 with RT-PCR was evaluated on coelomocytes. A significant change was found in enzyme activity and in the expression of the HSP70 gene and protein compared to the control. These results suggested that high-frequency stimuli elicit a noise-induced physiological stress response in A. lixula, confirming the vulnerability of this species to acoustic exposure. Furthermore, these findings provide the first evidence that cell-free coelomic fluid can be used as a signal to evaluate noise exposure in marine invertebrates.

Individual Sea Urchin Coelomocytes Undergo Somatic Immune Gene Diversification.

The adaptive immune response in jawed vertebrates is marked by the ability to diversify somatically specific immune receptor genes. Somatic recombination and hypermutation of gene segments are used to generate extensive repertoires of T and B cell receptors. In contrast, jawless vertebrates utilize a distinct diversification system based on copy choice to assemble their variable lymphocyte receptors. To date, very little evidence for somatic immune gene diversification has been reported in invertebrate species. Here we show that the SpTransformer (SpTrf ; formerly Sp185/333) immune effector gene family members from individual coelomocytes from purple sea urchins undergo somatic diversification by means of gene deletions, duplications, and acquisitions of single nucleotide polymorphisms. While sperm cells from an individual sea urchin have identical SpTrf gene repertoires, single cells from two distinct coelomocyte subpopulations from the same sea urchin exhibit significant variation in the SpTrf gene repertoires. Moreover, the highly diverse gene sequences derived from single coelomocytes are all in-frame, suggesting that an unknown mechanism(s) driving these somatic changes involve stringent selection or correction processes for expression of productive SpTrf transcripts. Together, our findings infer somatic immune gene diversification strategy in an invertebrate.

Analysis of oxidative stress and cellular aggregation in the coelomocytes of earthworms collected from metal contaminated sites of industrial and agricultural soils of West Bengal, India.

Endogeic earthworm Metaphire posthuma (Valliant, 1868) is a common biological component of the tropical soil of India and other countries. The species is reported to influence fertility and porosity of soil and bear a high composting potential. Intensive agricultural, industrial, and mining activities increase the amount of toxic metals in soil causing physiological adversity in earthworm and other biotic components in soil. Coelomocytes, the chief immunoeffector cells of earthworm, perform diverse physiological functions under the challenge of toxins and pathogens. The experimental earthworms collected separately from soils with agricultural and tannery activities were subjected to quantitation of prooxidation and antioxidation parameters for estimation of oxidative stress. Total count, cellular aggregation, generation of reactive oxygen species (ROS), superoxide anion, nitric oxide, activities of phenoloxidase, superoxide dismutase, catalase and glutathione-s-transferase, and amount of total protein were estimated in the coelomocytes of M. posthuma as experimental end points of toxicity screening. Concentrations of cadmium, chromium, lead, and mercury were determined in the soil samples to assess the degree of toxic contamination. The increase in the amount of prooxidants and decrease in the activities of antioxidant enzymes indicated the signs of oxidative stress in the coelomocytes of the organism. Aggregation of circulating coelomocytes is considered as an immune response involved in pathogen encapsulation response as reported in many invertebrates. Decrease in coelomocyte aggregation in earthworm collected from contaminated sites suggested a state of inappropriate shift of the innate immune status. Toxin-induced oxidative stress and reductions in cell aggregation response are the signs of immunocompromisation of M. posthuma. Present findings bear a prospect of this experimental species as an indicator of soil pollution.

Biomarker responses in earthworm coelomocyte extract - Noninvasively collected sample for pesticide effect assessment.

Earthworms are often used as model organisms in ecotoxicological research because of their natural habitat where they can be exposed to many different pollutants, including pesticides. Since a number of them has to be sacrificed for sample collection, it would be useful to develop non-invasive methods and techniques suitable for the analysis of target parameters. The aim of this study is to determine whether the coelomocyte extract, obtained by the non-invasive method, can be used to measure responses of biochemical biomarkers and to establish if it can be used in assessing the effects of pesticides already known to have a negative impact on the earthworms. In the present study Eisenia andrei earthworms were exposed for 48 h to organophosphates dimethoate and pirimiphos-methyl using the filter paper contact test. Following exposure, coelomocyte extracts were prepared and acetylcholinesterase (AChE) and carboxylesterase (CES) activities were measured. The percentage of inhibition of the measured enzymes in the coelomocyte extract was compared with the inhibition of the same enzyme activities in the samples obtained from the whole body homogenate. AChE and CES inhibition was observed at all concentrations for both pesticides in different types of samples. Compared to the coelomocyte extract, the level of AChE inhibition was slightly stronger in the whole body homogenate. Inhibition of CES at the same concentrations in different types of samples did not always coincide, especially in the case of dimethoate, however significant inhibition of CES in coelomocyte extract was recorded. This study indicates the possibility of using the coelomocyte extract for measurement of biochemical biomarkers and assessment of pesticide effects.

First report of Coelomomyces santabrancae sp. nov. (Blastocladiomycetes: Blastocladiales) infecting mosquito larvae (Diptera: Culicidae) in central Brazil.

A project from 2013 to 2017 sought to discover pathogenic fungi and oomycetes from dipteran species that are vectors of major diseases of humans and animals in central Brazil and to begin evaluating the potential of these pathogens as potential biological control agents concentrated on mosquito larvae. Some collecting sites proved to be especially productive for pathogens of naturally occurring mosquito species and for placements of healthy sentinel larvae of Aedes aegypti in various sorts of containers in a gallery forest in the Santa Branca Ecoturismo Private Reserve of Natural Patrimony (RPPN) near Terezópolis de Goiás (GO). Collections during May-April of 2016 and February 2017 yielded a few dead mosquito larvae of an undetermined Onirion sp. (Culicidae: Sabethini) whose hemocoels contained many ovoid, thick-walled, yellow-golden to golden-brown, ovoid thick-walled resistant sporangia, 38.3±4×22.8±2.3µm, decorated by numerous, closely and randomly spaced punctations of variable size and shape. These were the first indisputable collections from Brazil of any Coelomomyces species. Comparisons of the morphology of these sporangia with those of other species of Coelomomyces, confirmed that this Brazilian fungus represented a new species that is described here as Coelomomyces santabrancae.

Metallothionein 2 and Heat Shock Protein 72 Protect Allolobophora chlorotica from Cadmium But Not Nickel or Copper Exposure: Body Malformation and Coelomocyte Functioning.

Earthworms serve as good indicators of heavy metal contamination due to their innate sensitivity towards soil pollution. However, to date, not many studies have focused on endogeic earthworms, such as the omnipresent Allolobophora chlorotica. The current study was designed to verify whether this earthworm could serve as a novel distinctively susceptible species for environmental contamination studies. We show that the dermal exposure to Cu, Ni, and Cd affected the mortality and morphology of A. chlorotica, and the number and functioning of coelomocytes. These features particularly were pronounced in animals treated with Ni and Cu and interestingly to a lesser extend with Cd. In contrast, Cd induced a strong expression of metallothioneins (MT-2) and heat shock proteins (HSP72). The presence of MT-2 was detected not only in coelomocytes but also in the intestine, blood vessels, and epidermis. In conclusion, Allolobophora chlorotica coelomocytes are adopted to respond differentially to various heavy metals, generating powerful response towards potentially most dangerous exogenous non-essential elements.

Chronic endophthalmitis due to Pyrenocheta romeroi in an immunocompetent host--a case report from southern India.

PURPOSE: Endophthalmitis due to Pyrenochaeta romeroi has not been reported in literature (PubMed, Medline). We report an interesting case of P. romeroi causing chronic endophthalmitis in an immunocompetent lady. METHODS: Retrospective interventional case report. A 25-year-old immunocompetent lady presented with pain and redness in the left eye of 1-month duration. Her best-corrected visual acuity was 6/6 and 6/18 in the right and the left eyes, respectively. Slit-lamp examination of the left eye showed a corneal stromal scar, fibrinlike material in the anterior chamber, few retrolental cells, and normal fundus examination. RESULTS: Aqueous taps on two occasions were negative for bacteria and fungi on routine smear, culture, and nested polymerase chain reaction. As inflammation recurred despite intravitreal voriconazole and amikacin injections, a lensectomy with vitrectomy was done. During vitrectomy, dense flocculent material was seen in the pars plana with only scleral indentation. The flocculent material grew a rare filamentous fungus called P. romeroi. The left eye underwent retinal detachment surgery with silicone oil insertion for a giant retinal tear at 2 months of follow-up. At 6 months of follow-up, her vision in the left eye was stable at 6/24 (Snellen) with no ocular inflammation. CONCLUSION: P. romeroi may need to be added in the list of rare fungi, which cause chronic endophthalmitis.

[Subcutaneous infection spread by Scytalidium (Neoscytalidium) dimidiatum]. / Infection sous-cutanée disséminée par Scytalidium (Neoscytalidium) dimidiatum.

INTRODUCTION: S. dimidiatum (recently reclassified as N. dimidiatum) is a fungus that causes nail and/or superficial skin infection. It may also cause subcutaneous and deep infection, chiefly in immunocompromised patients. CASE REPORT: An 87-year-old male treated with oral corticosteroids for sarcoidosis consulted for violaceous cutaneous nodules on the back of his hands. Histopathological examination revealed epithelioid cell granulomas with numerous mycelial filaments and multiple spores. Culture of a biopsy sample resulted in growth of numerous colonies of S. dimidiatum and the patient was treated with intravenous amphotericin B. DISCUSSION: This organism is transmitted by direct or indirect contact with contaminated soil or plants. It mainly causes superficial skin and nail infections, and may result in deeper infections on rare occasions. We report a case of subcutaneous infection with S. dimidiatum in an immunocompromised patient (due to general steroid therapy) that was successfully treated using amphotericin B.

Atividade antifúngica de extratos de Momordica charantia L. sobre Colletotrichum musae / Antifungal activity of Momordica charantia L. extracts against Colletotrichum musae

Os objetivos do presente trabalho foram avaliar os efeitos de extratos de Momordica charantia sobre o crescimento micelial e a germinação de conídios de Colletotrichum musae, e a eficiência destes extratos no controle da antracnose, causada por C. musae, em bananas. Extratos aquoso e hidroetanólico, obtidos de folhas e ramos, na concentração de 50 por cento em relação ao volume adicionado, em meio sólido, proporcionaram 71 e 65 por cento de inibição do crescimento micelial, respectivamente, enquanto que em meio líquido, a inibição do crescimento micelial foi de 86 e 81 por cento, respectivamente. Somente o extrato aquoso e o tiofanato metílico, nas concentrações de 50 por cento e 1000 µg mL-1 respectivamente, proporcionaram 100 por cento de inibição da germinação de esporos de C. musae. Os extratos metanólico e aquoso inibiram em 80 e 70 por cento, respectivamente, o desenvolvimento das lesões em bananas, quando aplicados até dois dias antes da inoculação do fungo. Estes resultados foram semelhantes ao tratamento com tiofanato metílico, que inibiu 80 por cento do desenvolvimento das lesões. Confirma-se a presença de substância antifúngica nos extratos de M. charantia e outros estudos devem ser realizados para viabilizar seu uso no controle da antracnose da banana.

The aims of the present work were to evaluate the effects of Momordica charantia extracts on mycelial growth and conidial germination of Colletotrichum musae, as well as the efficiency of these extracts in controlling anthracnose caused by C. musae in bananas. Water and hydroethanol extracts were obtained from leaves and branches at 50 percent concentration relative to the added volume. In solid medium, extracts led to 71 and 65 percent mycelial growth inhibition, respectively, whereas in liquid medium the mycelial growth was inhibited at 86 and 81 percent, respectively. Only water extract and thiophanate-methyl, at 50 percent and 1000 µg mL-1, respectively, resulted in 100 percent inhibition of C. musae spore germination. Methanol and water extracts inhibited by 80 and 70 percent, respectively, the development of lesions in bananas when applied until two days before fungal inoculation. These results were similar to those of the treatment with thiophanate-methyl, which inhibited 80 percent development of lesions. The presence of antifungal substance was confirmed in M. charantia extracts. Future studies must be performed to make its use viable for the control of anthracnose in bananas.

Bilateral thalamic necrosis following ingestion of ridge gourd infested with coelomycete fungi (Diplodia).

Bilateral thalamic lesions detected on magnetic resonance imaging have a wide differential diagnosis. This report describes a previously healthy young man who developed bilateral thalamic necrosis with seizures, vomiting, hepatitis, neutrophilic leukocytosis and metabolic acidosis following consumption of raw dried fruits of the ridge gourd plant (Luffa acutangula) prescribed by a traditional medicine practitioner. These fruits were subsequently shown to be infested with spores and conidiomata of Diplodia, a coelomycete fungus known to cause neurotoxicity in farm animals. The patient made a partial recovery with supportive care, and has persistent deficits consistent with bilateral medial thalamic damage. This is the first report of neurological toxicity attributable to Diplodia in humans, and this entity should be considered in the differential diagnosis of bilateral thalamic lesions in the appropriate clinical setting.

The life history and in vivo culture of Coelomomyces utahensis (Blastocladiomycetes).

Coelomomyces utahensis is a fungal parasite of several genera of mosquitoes that inhabit rock-pools in southern Utah. Studies of the biology of Coelomomyces and their potential use in biological control of mosquitoes have been hindered by their complex life history, lack of axenic culture methods, and logistical problems producing their arthropod hosts for in vivo culture. In the case of C. utahensis, we have identified the alternate microcrustacean host as Potamocypris unicaudata, which is an ostracod that can be easily reared in abundance and stored for long periods. Described here are the life cycle and culturing of C. utahensis.